This competitive renewal application has two related parts aimed at resolving epigenetic mechanisms and chromatin features in B cell gene control. One line of studies is directed at defining the role of novel DNA methylation at CCWGG (i.e., CmCAITGG) sites in turning off B cell gene expression in lymphocyte development and in initiating chromatin modifications (histone deacetylation and CpG methylation) in gene silencing. We recently discovered CCWGG methylation in silenced B29 genes in AIDS-related Primary Effussion Lymphoma (PEL) and myeloma cells. We also showed that CCWGG methylation blocked critical transcription factor binding and repressed transcription of the B29 promoter. We will directly test the proposition that CCWGG methylation has two related regulatory functions: 1) to turn off gene transcription, and 2) to mark genes for chromatin modifications leading to gene silencing. CCWGG methylation and chromatin modifications in gene silencing will be analyzed in B cell genes (X5, V-pre-B, mb-i) that are turned off in development. The role of CCWGG methylation in gene silencing also will be tested using chromatin integrated CCWGG methylated vs. unmethylated B29 promoter-containing transgenes. A complementary line of studies will characterize the newly identified insulator located upstream of the B29 promoter and analyze its role in controlling B cell specific B29 expression. Insulators are recently identified boundary elements that flank genes and act as barriers to external influences that could affect gene expression (i.e., silencing, activation). The B29 insulator appears to be a new type of boundary element. Its location between the B29 gene and the upstream locus control region (LCR) that regulates expression of the pituitary growth hormone gene (located downstream of 829) predicts that it shields the B29 gene from inappropriate activation/expression. We will test this predicted activity of the B29 insulator using recombinase mediated cassette exchange (RMCE) to efficiently integrate transgenes into chromatin loci. The state of the B29 insulator also will be analyzed in B29-expressing vs. silenced cells to define the role of this element in gene silencing. These studies on CCWGG methylation and insulator function address new mechanisms in B cell gene control that supercede the effects of local trans-acting factors that interact with promoters and enhancers.